Since its conception more than 27 years ago, the principle of particle counting and sizing invented by Wallace H. Coulter has resulted in numerous improved methods and apparatuses for the electronic counting, sizing and analysis of microscopic particles, which are scanned in a fluid suspension of electrolyte, the first of which is shown by the pioneer U.S. Pat. No. 2,656,508 to Coulter. In this prior art particle analyzer, a D.C. electric current flow is established between two vessels by suspending electrodes in the respective bodies of the suspension fluid. The only fluid connection between the two bodies is through a microscopic orifice; hence, an electric current flow and field are established in and proximate to the orifice. The orifice and the resultant electric field in and around it constitute a sensing zone. As each particle passes through the sensing zone, for the duration of the passage, the electrical impedance of the contents of the sensing zone will change, thereby modulating the electric current flow and electric field in the sensing zone, and hence causing the generation of a signal to be applied to a detector suitably arranged to respond to such change.
In the commercial apparatus constructed in accordance with the heretofore mentioned U.S. Pat. No. 2,656,508, field excitation has been supplied by a direct current or low frequency source. The electrical change, i.e., D.C. signal, caused by the passage of a particle through the electric field of small dimensions, excited by a direct or low frequency current, is approximately proportional to particle size. A direct current is considered to be of zero frequency in this application. However, the impedance sensing principle has been expanded materially to provide information concerning particles being studied, not limited only to characteristics due to the size of particles, but including characteristics due to the composition and nature of the material constituting the particles, as disclosed in U.S. Pat. No. 3,502,974 to Coulter et al. and U.S. Pat. No. 3,502,973 to Coulter et al. These prior art apparatuses generally have at least two current sources, both of which are applied to the sensing zone simultaneously, one having a radio frequency (RF) and the other being the previously described "zero frequency" direct current (DC) or, alternatively, having a sufficiently low frequency that the reactive part of the particle impedance has a neglible effect on the response of the apparatus. One of the useful particle descriptors that can be obtained from this dual source arrangement is the "internal conductivity" or "opacity" of the particles. More specifically, with biological cells, their membranes have a very high resistivity in the range of a dielectric; however, the internal portion of the cell is fairly conductive. The RF current passes through the cell's membrane, thereby generating a detectable RF signal which correlates to the size, shape and conductivity of each particle. When the D.C. size signal for a cell is divided into the RF signal for that cell, a measurement termed "opacity" of the cell is obtained.
With the above described particle analyzers, the size and opacity measurements generally do not correlate exactly with the actual or true volume and internal electrical conductivity, respectively, of the cell. In apparatuses having hydrodynamic focusing, elongated particles will be aligned with their longitudinal axis substantially parallel to the axis of the sensing zone. With two equal volume particles, one being spherical and one being elongated, the spherical particle, while passing through the orifice, will have a greater cross section perpendicular to the current flow than the elongated particle. Hence, the spherical particle will distort the field in such a manner that it will give a greater measured size signal than the elongated particle, despite their equal volumes. Consequently, particles have been classified as to their shape by a term called "shape factor" which is used to correct their measured D.C. size signal. For instance, if an extremely elongated particle is assigned a shape factor of 1.0, then the spherical particle of the same volume has a shape factor of 1.5.
To correct for the inaccuracies introduced into the measured parameters by the particle's shape, the shape factor can be accurately measured on a cell by cell basis by obtaining a third signal, such as length, in addition to the RF and DC signals, and then correcting the measured parameters to obtain accurate values for the volume and internal conductivity of the cell, as described in U.S. Pat. No. 4,298,836, to Groves et al. However, this arrangement has the disadvantage of requiring an optical source and detector for obtaining the required length by making a "time of flight", i.e., length, measurement.
A drawback of the prior art electronic volume sensing particle analyzers is that slit scanning of the individual cell cannot be accomplished, such scanning being possible only with optical particle analyzers, as shown in U.S. Pat. No. 3,657,537 to Wheeless. More specifically, with the optical particle analyzers, a narrow illuminating beam, having a width less than the length of the cell traversing the same, excites fluorescence from a stained cell. In this manner the internal constituents of the cell are examined, such as the relative sizes of the cell's nucleus and cytoplasm. It is known that there are differences in internal conductivities of different portions of the cell, such as, for example, between the nucleus and the surrounding cytoplasm. However, in these prior art analyzers, these internal differences have not been measurable or subject to being quantified, due to the sensing zone created by the electric field of the sensing zone being always much longer in length than the cell. For instance, a sensing zone in a 75 micron long aperture will be substantially longer than the 75 microns and will frequently receive cells, such as red blood cells, having lengths in the range of 12 microns.
U.S. Pat. No. 3,720,470 to Berkhan discloses a flow chamber arrangement for moving particles along one side of the flow chamber's wall and PCT/EP80/0021 to Lindmo et al. discloses an arrangement for moving particles along the surface of a substrate. Previously mentioned U.S. Pat. No. 3,502,973 shows electrodes which create a wide electrical field that traverses the particle path.
The above described U.S. Pat. Nos. 3,502,973; 3,502,974; and 4,298,836 are incorporated by specific reference herein.